Biospecific multiparameter assay method

ABSTRACT

The object of this invention is an improved method for biospecific multiparameter assay method based on the use of different categories of microparticles as solid support for different bioaffinity reagents. This invention allows the use of microparticles of small size and with very moderate monodispersity and conventional short decay time fluorescent labels for labelling the biospecific reactants. The high sensitivity of this method is based on the use of confocal excitation and detection, or alternatively, two-photon excitation for measurement of the biospecific reaction. The identification of the category of the microparticle is based on the use of fluorescent or Raman scattering indicators associated with the microparticles representing different analytes.

This invention relates to a biospecific multiparameter assay method.Immunoassays are a well established group of biospecific assays and nowwidely used in routine diagnostics and research laboratories. Anothergroup of biospecific assays, is DNA hybridization assays. Biospecificassays generally employ one or several biospecific reactants (e.g.antibody, DNA probe) and normally one of these reactants is labelled.The labels currently used are radioisotopic, enzymatic, luminescent andfluorescent labels.

In routine diagnostics there is a growing need for multiparameter(multianalyte) analysis. Unfortunately, the current methodology does notallow the use of more than two or three simultaneous labels because thespectrometric signals from different labels cannot be sufficientlyseparated. The emission spectra of different radioisotopic labels andphotoluminescent labels overlap significantly and as a consequence theyprovide inadequate separation of different analytes over a requiredconcentration range.

The purpose of this invention is to present a better method formultiparameter biospecific assays. The method according to thisinvention is based on methods that are generally known within the fieldof immunology and DNA hybridization. Normally, they are performed asfollows. The method uses two biospecific probes that recognize theanalyte molecule k. In this text, these probes are referred to as theprimary probe Ab(k,1) and the secondary probe Ab(k,2). When thesecondary probe is labeled, for example, with a photoluminescent labelF, it is denoted with the symbol Ab^(F) (k,2). In the reaction solution,there is an excess of primary and secondary probes compared to thenumber of analyte molecules M_(k). When the analyte molecule, which iseither a polypeptide or a macromolecule, has separate epitopes i.e.molecule structures that bind specifically to the probes, they formtogether a complex Ab(k,1)+M_(k) +Ab^(F) (k,2). In principle, the amountof complex formed is directly proportional to the amount of the analyte,and the excess of primary and secondary probes remain in the solution.The complexes are separated from the free probes using a commonly knowntechnique, for example, in which the primary probe is bound to a solidcarrier and the free probes are washed away from the sample. Finally,the signal of bound label F in the complexes is measured in atraditional way which depends on the label chosen. The intensity of thesignal obtained is directly proportional to the amount of label in thesolution, and the response of the system is linear.

If the analyte to be measured is a small molecule without two or moreepitopes which specifically bind to the probes, one can use a secondaryprobe that reacts specifically with the complex formed by the analyteand the primary probe (C. H. Self et al., Clin. Chem. 40 (1994)2035-2041).

BACKGROUND OF THE INVENTION

Certain multiparameter biospecific assay methods have been introducedearlier. It has been common practice to use multiple labels to labelbiospecific reagents and to perform the separation of the signals on thebasis of their different emission spectra. In most cases, however, theknown multiparameter methods are based on the use of a solid supportwhere the biospecific reagents can be immobilized at separate andoptically distinguishable areas, or that are based on the use ofartificial microparticles as a solid support. Some of the methods arereviewed below:

1. A method, in which various biospecific probes are attached to amatrix, which is formed by small areas on a planar solid support, isdescribed in the patent PCT WO 84/01031. In this method, after thereaction and the wash, the signals from the photoluminescent labels ineach area are measured separately, for example, using a laser scanningmicroscope.

2. A method, in which the identification of the analyte category isbased on the color of the microparticles, which are used as a solidsupport and which is achieved by optically measuring the lightabsorption of the particle to be analyzed (J. G. Streefkerk et al.Protides Biol. Fluids 24 (1976) 811-814 and U.S. Pat. No. 5,162,863).

3. A method, in which the identification of the analyte category isperformed by optically measuring the absorption of the dye inside theparticle, the refractive index or the size of the particle to beanalyzed (U.S. Pat. No. 5,162,863).

4. A method, in which the identification of the analyte category isbased on the use of different particle sizes and in which theidentification is performed by optically measuring the diameter of theparticle to be analyzed (T. M. McHugh et al., Journal of ImmunologicalMethods 95 (1986) 57-61).

5. A method, in which the microparticles are identified by means offluorescent dyes that are mixed or impregnated within the particles, andthe biospecific signal is measured from the fluorescence intensity ofanother fluorescent dye, such as FITC (EP 126450, GOIN 33/58).

6. A method, in which a dye emitting short decay time fluorescence(decay time a few nanoseconds), is used for the identification ofmicroparticles, and a dye emitting long decay time fluorescence (decaytime from 10 microseconds to 2 milliseconds), is used for measuring theanalyte concentrations, and in which a time resolved fluorometer is usedfor the discrimination of the short and long life time fluorescence(U.S. Pat. No. 5,028,545).

7. A method, in which a dye emitting short decay time fluorescence(decay time a few nanoseconds), is used for the identification of themicroparticles, and a molecule which generates chemiluminescence orbioluminescence (decay time several seconds), is used to measure theanalyte concentrations, and in which the fluorescence and luminescentsignal can effectively be separated from the fluorescence because theyare excited and they emit light at different times (FI-patent 89837).

8. A method, in which a dye emitting short decay time fluorescence(decay time a few nanoseconds), is used for the identification of themicroparticles and a dye emitting phosphorescence, (decay time from 10microseconds to 2 milliseconds), is used to measure the analyteconcentrations, and in which a time resolved fluorometer is used for thediscrimination between the short decay time fluorescence and the longdecay time phosphorescence (FI-patent 90695).

9. A method, in which dyes emitting long decay time fluorescence, suchas fluorescent chelates of lanthanide ions Tb, Dy, Eu and Sm, are usedfor the identification of the microparticles and for measurement of thebiospecific signal (FI-patent application 931198).

A common problem in many multiparameter assays mentioned above is thatthe signal of photoluminescent label, which indicates the analytecategory, and the signal from the photoluminescent label, which measuresthe concentration of the biospecific probe, interfere with each other.This is a problem that significantly restricts the dynamic range of themeasurement of the analyte concentration. This interference may becomeparticularly significant when measuring low analyte concentrations andwhen a wide dynamic range is required for the measurement of thebiospecific signal. In methods 6, 7, and 8 referred to above,interference is eliminated by choosing such photoluminescent labels forthe measurement of the biospecific reaction and identification labelswhich have substantially different emission decay times. In methods 1,2, 3 and 4 the analyte is identified using an alternative method ratherthan using a photoluminescent label. In methods 5 and 9, theidentification method of the analyte essentially restricts the dynamicrange of the measurement.

Another problem associated with methods 6, 7, 8 and 9 mentioned above isthe long measurement time, caused by the long decay time (T1/2=1millisecond) of the fluorescent and phosphorescent labels. This problemis caused by the saturation of the excited states of the labels, whichrestricts the intensity of the exciting light to such a low level that ameasurement time of up to one second is needed for each microparticle.Likewise, the measurement of the signals from labels that are based onchemiluminescence, bioluminescence and electroluminescence, also take atleast one second.

OBJECTS OF THE INVENTION

The object of this invention is an improved method for biospecificmultiparameter assay method based on the use of small microparticleswith a diameter range from 100 nanometer to 10 micrometer, but notlimited to these measures. The microparticles are used as solid supportfor different bioaffinity reagents in reaction solution to which thesample is added. This invention allows the use of microparticles withvery moderate monodispersity. The method of this invention allows theuse of conventional short decay time fluorescent labels for labellingthe biospecific reactants. Despite the fluorescent background, which isnormally associated with such fluorescent labels, the opticalmeasurement method of this invention ensures an ultimate sensitivitywhich potentially can be one molecule per microparticle.

The following terms are used systematically later in the text: The term"indicator" is used in the context of a substance used foridentification of the microparticles. The term "dye" will be used if theindicator is a fluorescent dye. The term "label" will be used in thecontext of labelling of the biospecific reactant with a photoluminescentlabel F, which is a fluorescent dye with short decay time.

In particular this invention relates to the method of sensitivedetection of the signal from the label F and identification of thecategory of microparticles using indicator D_(k).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a functional diagram of the measuring system needed for themethod of this invention.

FIGS. 2a-2j show the excitation and emission spectra of thetetrapyrrolic compounds, which can be used as indicator D_(k).

FIGS. 3a and 3b show the excitation and emission spectra of selectedtetrapyrrolic compounds which can be used as indicator D_(k).

FIG. 4 presents an example of signals derived from the detector.

FIG. 5 shows the single photon count rate as a function of the number ofmolecules in the detector focal volume.

FIG. 6 shows a functional diagram of an alternative measuring system.

FIG. 7 shows a functional diagram of an alternative measuring system.

DETAILED DESCRIPTION OF THE INVENTION

In the method of this invention, the microparticles are in differentcategories representing different analytes to be assayed, saidcategories comprising different amounts of one or several indicators D₁,D₂, D₃, . . . D_(k) (later D_(k)), for the purpose of identification ofthe category of said microparticles. Each category of microparticles iscoated with a different biospecific reactant Ab(k,1), which reacts withthe particular analyte M_(k) and with a secondary reagent Ab^(F) (k,2)and which is labelled with a photoluminescent label F. ComplexesAb(k,1)+M_(k) +Ab^(F) (k,2) are formed in the reaction on the surface ofthe microparticles belonging to category k and the signal from the labelF is then measured and used for determination of the concentration ofeach analyte M_(k) in the reaction solution.

Said method is comprised of steps of:

pooling the different categories of microparticles coated with theprimary reagent Ab(k,1) together in a suspension and adding the sample Mcontaining analytes M₁, M₂, . . . M_(k) to be assayed into thesuspension,

adding a mixture of labelled biospecific secondary reactants Ab^(F)(k,2) into the suspension to initiate biospecific reactions between theanalytes M_(k) and the labelled reactants and microparticle-associatedreactants Ab(k,1),

diluting the suspension to reduce the concentration of labelledreactants not bound to the microparticles,

sampling the suspension in order to expose one microparticle at a timeto a laser beam and exciting the indicators D_(k) and thephotoluminescent label F associated with the microparticle,

converting the signal obtained from the excited indicators D_(k) andfrom the label F to electrical signals,

identifying the category of each microparticle on the basis of theelectrical signal resulting from the indicator D_(k),

determining the concentration of the analyte M_(k) on each microparticleon the basis of the electrical signal resulting from the photonemissions generated by the label F.

A sufficient number of microparticles is analyzed and the result of eachmicroparticle is registered in a computer.

The suspension is diluted adequately after the reaction. Often adilution of one order of magnitude may be enough for sufficientseparation of the bound and free fraction, because the photometricdetector used in this invention is able to discriminate opticallybetween those signals originating from the microparticle within theoptical focal volume and the signal from the free label in thesurrounding buffer solutions.

The photoluminescent label F in the method of this invention, is a shortdecay fluorescent compound and the sample is illuminated by a continuousor pulsed laser beam which excites the fluorescence emissions. Thefluorescent label, with a typical emission decay time of a fewnanoseconds, allows one to use an intensity of the exciting light up to10⁶ times higher than that of the excitation intensity of the long decaytime fluorescent labels in the method referred to above. A much strongersignal with a short decay time can be derived from labels and,correspondingly, the signal can be measured precisely in much shortertime. Since the time needed for detecting one microparticle is veryshort, for example 100 microseconds, the method of this invention hasthe advantage that a large number of microparticles can be measuredwithin a short time. This results in higher capacity, accuracy andprecision.

The background signal, which seriously reduces the sensitivity ofordinary fluorometry, is eliminated in this invention by using confocalexcitation and detection or alternatively, by using two-photonexcitation. Both of these methods restrict the active volume ofmeasurement to a diffraction limited volume which approximatelycorresponds to the volume of one microparticle. Both of these methodsand the related optical systems discriminate very efficiently allbackground scattering and fluorescence which originate from outside theactive diffraction limited sample volume.

It is essential for the sensitivity of the method of this invention, aswell as for the sensitivity of the other multiparameter assayspreviously known and referred to above, that the signal from theindicator used for the identification of the analyte, does not interferewith the signal from the photoluminescent label used for the measurementof the biospecific reaction. In this invention, this interference hasbeen eliminated by using identification with nonfluorescent indicatorsor alternatively, when using a fluorescent indicator, the interferenceis eliminated with the use of the two-photon excitation method.

Realization of the Invention with the Confocal Principle

The confocal principle applied to this invention is described infollowing with the reference to FIG. 1, which shows an example of thefunctional diagram of the measuring system needed for the method of thisinvention. The laser (1) is used for excitation of fluorescence and itis focused through a lens (2a) and an objective lens (2b) to the sample(3) in a capillary cuvette (4). The fluorescence signal from the sampleis directed by a dichoic mirror (5) to a pinhole (6). The light beamfrom the pinhole (6) is spit by dichroic mirrors (7) and (8) passes tothe detectors (9), (10) and (11), which are tuned for the wavelengths oflabel F and indicators D₁ and D₂ respectively. For simplicity, thefunctional diagram presented now is applied to the registration of onlytwo spectral features of the indicators D_(k) requiring two detectors(10) and (11), respectively. The detectors incorporate appropriatespectral filters. The detectors (9), (10) and (11) are connected to asignal processor (12). The signal processor converts the signals tonumerical form and the results are processed in the computer (13), whichalso controls the hardware. The label F and the indicator D_(k) can alsobe excited with different lasers, if their excitation wavelengths aredifferent, or if better results can be achieved by using two lasers. Inthis case, both lasers are focused to the same or adjacent points of themoving sample.

The principle of the confocal set-up is described below with referenceto FIG.1. Firstly, imaging of a point-like light source (1) to the focalplane (3) of the objective lens (2b) is described. Due to diffraction, apoint-like source of light forms an intensity distribution, which ischaracteristic to the optical system, in the focal plane. The intensitydistribution is called the point spread function, which is determined inthree dimensions. A normalized point spread function defines theprobability S1 of how photons, radiated from a point-like source, aredistributed on the focal area (3); that is, the probability that thephotons are absorbed to different parts of the sample volume.

A corresponding point spread function S2 can also be determined for thespatial distribution of the photons emitted from the focal point thatreach the pinhole in front of the detectors. The value of thisnormalized function in the vicinity of the focal point defines theprobability of the photons emitted from different points and hitting thepinhole (6).

In the confocal optical system that has been applied to the method andthe device of this invention, the light source (1) and the pinhole (6)are focused to the same focal point (3). The probability that a photonradiated from a point-like light source (1) causes a fluorescenceemission in the sample, and that the emitted photon hits the pinhole(6), is described by the normalized product S1*S2 of the illuminationand detection intensity distributions. The probability distribution thusderived, is three dimensional and is clearly more restricted than theone produced by conventional optics, especially in the axial direction.The fluid volume to be measured in a confocal system is considerablysmaller than the one in a conventional optical system. When using anobjective lens with a large numerical aperture (N.A.>0.5) and a confocalsystem, the active fluid volume is reduced to under a tenth of what isrequired in a conventional optical system. The dimension of the fluidvolume under observation, is clearly larger axially than laterally, andit is inversely proportional to the square of the numerical aperture(N.A.).

Realization of the Invention with two Photon Excitation

An alternative labelling and detection method that allows the use offluorescent dyes as the identification indicator D_(k) and offers a goodseparation between the signals from D_(k) and F, is based on two-photonexcitation of the label F.

Normally, the fluorescent labels are single-photon excited, which meansthat the chromophores of the fluorescent label absorb light at thewavelength of the exciting light beam. Two-photon excitation can replacesingle-photon excitation and reduce the background caused by scatteringand autofluorescence and it also very efficiently eliminates thespectral interference between D_(k) and F. Two-photon excitation ispossible when, by focusing an intensive light source, the density ofphotons per unit volume and per unit time becomes high enough for twophotons to be absorbed into the same chromophore. In this case, theabsorbed energy is the sum of the energies of the two photons. Alreadyin the 1930's, two-photon excitation of fluorescent materials wastheoretically known, and from the 1960's on it has been applied in thefields of spectroscopy and microscopy (U.S. Pat. No. 5,034,613).According to the concepts of probability, the absorption of a singlephoton by a chromophore, is an independent event, and the absorption ofseveral photons is a series of single, independent events. Theprobability of absorption of a single photon can be described as alinear function as long as the energy states that are to be excited arenot saturated. The absorption of two photons is a non-linear process ofthe second kind. In two-photon excitation, the chromophore is excitedonly when both photons are absorbed simultaneously, which isapproximately within a femtosecond. The probability of absorption of twophotons is equal to the product of probability distributions ofabsorption of the single photons. The intensity of fluorescenceemission, caused by two photons, is thus a quadratic process withrespect to the photon density of illumination.

The properties of this invention's optical system have been describedabove with respect to the response of the system to a point-like lightsource. A point-like light source forms, due to diffraction, anintensity distribution in the focal plane characteristic of the opticalsystem (point spread function). When normalized, this point spreadfunction is the probability distribution for the photons from the lightsource to reach the focal area. In two-photon excitation, theprobability distribution of excitation equals the normalized product ofthe intensity distributions of the two photons. The probabilitydistribution thus derived is 3-dimensional, and is clearly morerestricted than that for single-photon excitation, especially in thevertical direction. Thus in two-photon excitation, only the fluorescencethat is formed in the clearly restricted 3-dimensional vicinity of thefocal point is excited.

When a chromophore is two-photon excited and the excitation isrestricted to the 3-dimensional vicinity of the focal point, then thesignal caused by scattering in the vicinity of the focal point and fromthe optical components, is reduced remarkably if compared to normalexcitation. Furthermore, two-photon excitation decreases the backgroundfluorescence outside the focal point, in the surroundings of the sampleand in the optics. Since the exciting light beam must be focused onto assmall a point as possible, two-photon excitation best suits theobservation of small sample volumes and structures, which is also thesituation in the method according to this invention.

The previously mentioned advantages of two-photon excitation are basedon the fact that visible or near-infrared (NIR) light can be used forexcitation in the ultraviolet or blue region. Similarly, excitation inthe visible region can be achieved by NIR light. Because the wavelengthof the light source is considerably longer than the emission wavelengthof the chromophore, the scattering at a wavelength of the light sourceand the possible autofluorescence can be effectively attenuated by usinglow-pass filters (attenuation of at least 10 orders of magnitude) toprevent them from reaching the detector.

The common way to produce two-photon excitation is to use ultra-shortlaser pulses. During the short pulse, it is possible to achieve asufficiently high energy density for two-photon excitation, but theaverage energy is kept low. It has been observed, though, thattwo-photon excitations can also be observed with continuous-wave laserillumination.

In our experiments, we have observed that a very highsignal-to-background-ratio and good sensitivity can be reached withtwo-photon excitation and short-lived fluorescent labels. Suitablefluorescent labels for two-photon excitation are, for example, coumarin,rhodamine derivatives and phycobiliproteins.

In using the method of two-photon excitation, the coincidence conditionof the laser pulse and the pulse from the photon detector can also beused to eliminate thermal noise from the photon detector. In this case,thermal noise becomes insignificant. The use of two photon excitation isadvantageous compared to any single-photon excitation method becausescattering and background noise, especially that caused by proteins andother macromolecules in the sample, is considerably lower. Nofluorescence arises at the wavelength of the laser, nor can scatteringcaused by the laser beam reach the detector, because low-pass filterseffectively block wavelengths lower than that of the laser.

Two-photon excitation can best be performed with pulse lasers. The shorttransit time requires a pulsed laser with very high repetitionfrequency. Today, the laser suitable for this application is thetitanium-sapphire femtosecond laser with pulse energy of 10 nJ and withpulse frequency of 76 MHz and with adjustable 700-900 nm wavelength.Less expensive pulsed lasers suitable for this application will likelybe available in the near future. An example of this kind of developmentis the mode-locked 300 MHz pulsed diode laser (Laser Ionics Inc.,Orlando, Fla., USA), lambda=825 nm, pulse energy 0,03 nJ, pulse width1-20 ps. Another example is a new, not yet commercially available diodepumped CrLi-Sapphire laser with 80 MHz pulse rate, 30 fs pulse width,0.5 nJ pulse energy and adjustable 820-900 nm wavelength.

Two-photon excitation can provide a diffraction-limited focal volumewhich is slightly larger than that of the confocal set-up, but clearlydefined in three dimensions. The lower resolution is simply aconsequence of using a longer excitation wavelength for two-photonexcitation. However, the optical system for two-photon excitation canalso be combined with a confocal set-up. By choosing an appropriatedetector pinhole it is possible to optimize the size of the focalvolume.

Identification of the Microparticles with Raman Indicators

The use of Raman indicators is an alternative method for identificationof the microparticle category. This method is well suited both for theconfocal and two-photon excitation concept.

Raman scattering is inelastic light scattering from molecular structureswhere the energy due to molecular vibrations is mixed with the Ramanscattering due to light excitation. The Raman spectrum is expressed as awavelength shift on both sides of the wavelength of the incident light,and called the Stokes shift and the anti-Stokes shift. A Raman spectrumincludes information from the molecular structure similar to that froman infrared (IR) spectrum. Raman spectrum can also be obtained from verysmall objects like microparticles (P. Dhamelincourt & al. SpectroscopyEurope 5/2(1993) 16-26). Therefore, Raman spectra can be used toidentify and to distinguish different molecular structures ofmicroparticles.

Microparticles for different categories can be produced with knownpolymerization methods employing chemically different monomer mixturesfor different particle categories. The monomer mixtures can includedifferent kinds of monomers in different quantities or indicatorsubstances can be added to the monomer. Examples of different monomerssuitable for polymerisation and having significantly different Ramanspectra are styrene, deutered styrene (=styrene-D6), methylmethacrylateand acrylonitrile. Examples of additional indicators are oligomers orpolymers of above mentioned monomers or aliphatic or aromatic halogencompounds.

Microparticles with a diameter ranging from 100 nanometers to 10micrometers can be produced from different materials or materialcomposites. The microparticle indicator substances can be added toproduction batches of different categories of microparticles and indifferent combinations. Thus the Raman signals from different indicatorsexpress the category to which the microparticle belongs. For example,when using styrene as main component of the monomer mixture for particlesynthesis and when using the other three monomers mentioned above, asindicators, it is possible to produce microparticles in 2³ =8 differentcategories. The Raman spectra obtained from microparticles of thesedifferent categories can be recognized by comparing the spectra usingthe correlation method described e.g. in U.S. Pat. No. 5,313,406 or bycomparing the intensities of certain individual spectral peaks.

Identification of Microparticles with Fluorescent Indicators

The use of fluorescent dyes is an alternative method for identificationof a microparticle's category. This method is well suited only for thetwo-photon excitation concept because spectral overlap may causesignificant interference in the confocal concept.

Fluorescent microparticles with a diameter ranging from 100 manometersto 10 micrometers can be manufactured combining the polymer materialwith a suitable short decay time fluorescent dye. Well known fluorescentdyes with very short decay time, for example POPOP, bisMSB, fluoresceinor rhodamine etc., can be added to any monomer (as discussed e.g. in"Theory and Practice of Scintillation Counting", Ed. J. B. Birks,Pergamon Press, 1967, pp. 321-353) and solid fluorescent material isformed in polymerization. The material can be processed intomicroparticles in the same step. The fluorescent dyes can alternativelybe impregnated into the surface of the microparticles or coupled on thesurface of the microparticles. The fluorescent dye can be added intodifferent batches of the monomer in substantially differentconcentrations differing e.g. by a factor of two from each other.

Irrespective of the choice of the label F and excitation method, severalcompounds with short fluorescence decay time can be used as anidentification dye D_(k). It is clear that the measuring device neededin this method can be simplified, if the excitation wavelength for allof the chromophores, F and D_(k), is the same, and the emissionwavelengths differ substantially from each other, so that they caneasily be separated spectrally. It is important that the fluorescent dyechosen has an absorption band that does not overlap with the emissionband of the photoluminescent label F. The measuring device issimplified, if the chromophores F and D_(k) can both be excited with thesame light source.

In case of two-photon excitation, for example, each of the chromophores,F and D_(k), can be two-photon excited, or alternatively, F istwo-photon excited and the fluorescent dyes of the NIR region are usedas chromophores D_(k) and they are one-photon excited by the fundamentalwavelength of the same laser. However, if chromophores similar to theones described above are not used, it is necessary to excite them withtwo separate lasers, which are focused to the same or adjacent points ofthe moving sample. The excitation of the points, and the emission thatfollows it, are separated in the time domain.

The potential fluorescent chromophores D_(k) should show the followingproperties. In the method according to this invention: 1) They must havea common fluorescence excitation wavelength, which lies, if possible, onthe excitation range of the bioaffinity label F, or at the fundamentalwavelength of the laser that is used for the two-photon excitation of F;2) they must have spectrally separable fluorescence emission bands thatare higher than that of F; 3) the decay time of their fluorescenceexcited states must be short, and lie in the nanosecond range; inaddition it is advantageous for a good function of this invention if 4)they have a large difference between their excitation and emissionwavelengths; 5) they have no significant long decay emission component;6) they are chemically stable and attachable to the microparticles; and7) it would be very useful if their susceptibility to two-photonexcitation is lower than that of F.

The following commercially available and widely used dyes meet thesespecifications: Hoechst 33258, rhodamine (TRITC), Texas Red and QuantumRed. Although the excitation maxima of these dyes lie at differentwavelengths, they can still be reasonably well excited at a singlewavelength in the 300 to 450 nm range. The emission maxima lie atwavelengths of 470, 570, 620 and 670 nm, respectively.

A group of compounds suitable for dyes D_(k) can be found among thefollowing group of tetrapyrrols: porphyrins, chlorines,bacteriochlorines, purpurines, pheophorbides, phthalocyanines andnaphthalocyanines. These compounds generally have overlapping absorptionbands at a near-UV range (320 nm-450 nm, the so called "Sores band") butalso from the visible to the NIR-range and narrow separate fluorescenceemission bands at the red and NIR-range (600 nm-1200 nm). Thesecompounds can be produced synthetically or microbiologically(Porphyrins, D. Dolphin, Ed., Elsevier Amsterdam-N.-Y.-London, 1980, V.1-3) and they have been used in various analytical applications (D. B.Papkovsky, Appl. Fluor. Technology 3 (1991) 16-23; EP 0127797; EP0071991; Russian patent SU 1,659,477). Examples of tetrapyrrolic dyesare: 1) deuteroporphyrine IX, 2) mesoporphyrine IX, 3) proto-porphyrine(IX) dimethyl ester, 4) octaethylporphin, 5) tetraphenylporphin, 6)tetra-(2-metoxy)-phenyl-porphin, 7) chlorine of coproporphyrin dimethylester, 8) bacteriochlorine of coproporphyrin dimethyl ester, 9)aluminium phthalocyanine and 10) zinc phthalocyanine.

Another group of compounds suitable for dyes "D" can be found among thecyanine dyes. Examples of such cyanine dyes are:

3.3'-diethylthiatricarbocyanine perchlorate,

1.1'-diethyl-2.2'-dicarbocyanine iodide,

3.3'-diethyloxadicarbocyanine iodide,

3.3'-diethyloxatricarbocyanine iodide,

3.3'-diethylthiadicarbocyanine iodide,

3.3'-diethylthiatricarbocyanine iodide.

                                      TABLE 1    __________________________________________________________________________    Dye                   λexc       λem    __________________________________________________________________________    1 deuteroporphyrin IX 397                             496                                527                                   569      622                                               651                                                  689    2 mesoporphyrin IX    398                             498                                531                                   569      624                                               691    3 protoporphyrin IX, dimethylester                          402                             503                                538                                   516      633                                               671                                                  702    4 octaethylporphin    392                             496                                527                                   567      623                                               653                                                  689    5 tetraphenylporphin  416                             512                                548                                   593      654                                               717    6 tetra-(o-methoxy)-phenylporphin                          421                             518                                552                                   597      656                                               721    7 chlorin of coproporphyrin dimethyl ester                          377                             402                                492                                   541                                      590                                         640                                            650    8 bacteriochlorin of coproporphyrin dimethylester                          370                             468                                498                                   542                                      636   643                                               720    9 aluminium phthalocyanine                          345                             607                                636         679                                               748    10      zinc phthalocyanine 347                             538                                612         689    11      3.3'-diethylthiatricarbocyanine perchlorate                          772               818    12      1.1'-diethyl-2.2'-dicarbocyanine iodide    13      3.3'-diethyloxadicarbocyanine iodide                          578               605    14      3.3'-diethyloxatricarbocyanine iodide                          695               719    15      3.3'-diethylthiadicarbocyanine iodide                          662               679    16      3.3'-diethylthiatricarbocyanine iodide                          772               820    17      IR-125              795               833    18      IR-132              832               905    19      IR-140              826               882    20      IR-144              745               825    21      coumarin 120        334                             393            426    __________________________________________________________________________

Additional examples are commercial laser dyes such as IR-125, IR-132,IR-140 and IR-144 (Eastman Laboratory Chemicals, Catalog No. 55, Edition93-94)

The excitation (exc) and emission (em) wavelengths of the tetrapyrrolicand cyanines dyes listed above are presented in Table 1. The excitationand emission spectra of the compounds 1-10 are shown in FIG. 2a-2j. Itcan be seen that the spectra fulfil the requirements 1-4. Regardingrequirement 6, we have found that tetrapyrrolic dyes show very lowsusceptibility to two-photon excitation. Phycoerythrin, for example,shows very high susceptibility to two photon excitation. Because of thisfeature, tetrapyrrol dyes and phycoerythrin are a very good combinationas D_(k) and F.

These tetrapyrrolic and cyanine dyes can be supplemented with otherknown organic fluorescent dyes having the same excitation wavelength andshowing an emission band either within the lower or the higher side ofthe emission range of tetrapyrrolic or cyanine dyes. An example of thiskind of compound is coumarin 120 in Table 1.

It can be seen from the excitation and emission spectra of thetetrapyrrolic compounds 1-10 presented in FIG. 2a-2j that it is easy tofind at least three different compounds with separate emission spectra.As an example we show in FIG. 3b emission spectra for three compounds 1,6 and 10, their emission wavelengths being 623 nm, 656 nm and 689 nm,respectively. These compounds are shown together with the emissionspectrum of coumarin. The spectrum for each compound can be measuredover a minimum of one order of magnitude dynamic range without anysignificant interference by the other compounds when the concentrationsof the other compounds vary to same degree. The excitation spectra ofthese same compounds are presented in FIG.3a. All compound can beexcited in the wave length range of 350-400 nm. The compounds were in asolid matrix (Merckoglas). In FIG. 3b the excitation spectrum (solidline) and the emission spectrum (dotted line) are shown.

Microparticles having fluorescent dyes like tetrapyrrolic or cyaninedyes, either internally or on their surface, can be produced bydifferent known methods. The dyes can be attached to the particlesurface covalently if the particles are provided with chemically activegroups (Molday, R. S. et al., J. Cell. Biol., 64 (1975) 75-88). The dyecan also be impregnated within the surface layer of the microparticlesby swelling the particles first in an organic solvent and then bywashing and evaporating the solvent after impregnation. These methodshave been described e.g. in following manufacturer's publications:Dyeing Large Particles, published by The Dow Chemical Co., 1972 andUniform Latex Particles, published by Seragen Diagnostics Inc. Naturallythe dyes can be added to the raw material before polymerization of theparticle.

Signal Analysis

One possible design concept is to use single photon counters asdetectors (9) (10) and (11) in FIG. 1. The signals from these detectorsare characteristically single photon signals, which are transformed tobinary digital signals with a duration, for example, of 10 nanoseconds.FIG.4(A), 4(B) and 4(C) present examples of pulses derived from thedetectors as a function of time. A microparticle remains for a timet_(m), referred to as transit time, under the excitation of the laserbeam within the volume defined by the point spread function of theconfocal optics. In practice, the transit time depends on the speed ofthe flow inside the cuvette (3, FIG. 1), and is typically 100microseconds. The intensity of the laser beam, used for excitation oflabel F, can be set so high at the focal point (3) that it can nearlysaturate the excited states. If the decay time of the fluorescent labelsF is only about 1 nanosecond, the particles can be excited and relaxedup to 10⁵ times within the time interval of t_(m) =100 microsecondsunder the influence of a powerful laser beam. The number of photonsobserved by the detector, depends on the quantum efficiency of the labelF, the collection efficiency and the losses due to the optics and thequantum efficiency of the photon detector (9). In practice, a detectionefficiency of 10⁻² can be obtained using avalanche photon diode counterswith 80% quantum efficiency (EG&G Optoelectronics, Canada, typeSPCM-141-AQ). Within the transit time t_(m), the detector (9) can detectone or many photons from the fluorescence emission which come from onemicroparticle flowing through the active volume. The photons appear asstochastic photon bursts within the transit time t_(m) (section 1,FIG.4). In addition to these bursts, many other stochastic signals mayalso be detected (section 2, FIG.4). They originate from the backgroundfluorescence, from free molecules in the sample and from scattering andthermal noise.

An alternative photon detector is a photomultiplier tube and inparticular a hybrid photomultiplier tube, which is composed of a singlestage electron multiplier and a silicon diode as the electron sensor.This photomultiplier tube is capable of producing an analogue pulsewhich resolves a single photon. The advantage of the photomultipliertube is its high dynamic range but the disadvantage is a quantumefficiency one order of magnitude lower than that of avalanche photodiodes. It is useful to adjust the laser intensity to the optimalfrequency of single photons to be detected during the photon burst fromeach kind of sample to be analyzed. If the laser intensity is too high,the rate of photons exceeds the counting speed of the detector and thePoisson distribution of the counts in the time domain will be distortedand consequently the discrimination between true signal and noise is notoptimal when using auto-correlation analysis. The excitation power is anadjustable parameter for the optimal photon emission rate from thesamples in each particular application.

The avalanche diode photon counter may generate spontaneousafter-pulsing with the probability of 10⁻³. Avalanche photon counters,as well as photomultiplier tubes, suffer from thermal noise whichappears as stochastic counts. The afterpulsing and the noise can beeliminated with the following alternative methods. Auto-correlating thesignals form avalanche photon counters with a threshold of 3 counts orhigher eliminates the background caused by after-pulsing, but this ismade at the cost of detection efficiency. By dividing the emission beaminto two parts with a 50%/50% beam splitter for two separate photoncounters and using a cross-correlator, it is possible to discriminatethe afterpulses and thermal noise. The increased optical losses can becompensated with increased laser power.

The discrimination power between the background counts and the truecounts from the particles can be enhanced further by cross-correlatingthe signals obtained from detectors (9), (10) and (11).

The correlation analysis of the single photon counts can include bothauto-correlation analysis and cross-correlation analysis. Theauto-correlation analysis is based on registration of the time intervalsbetween the photon counts from each detector. Application of thecorrelation analysis for two or several independent photon detectors iscalled cross-correlation. The emitted photons from the true particles,detected by one or several detectors during the transit time of theparticle, can correlate in the time domain and within the followingcorrelation parameters: correlation time, correlation thresholds as aminimum number of counts per detector, coincidence threshold defined forthe condition of coincident counts from independent detectors. Thesecorrelation parameters are adjustable for optimal discrimination ofnon-specific photon counts.

The device (12) in FIG. 1 performing the correlation analysis for singlephoton bursts can be an electronic logic circuit, which gives an outputsignal if a pre-set number of single photon counts from each detectorarrives within a pre-set period of time. The circuit may also performmore complex correlation functions or the circuit may be replaced byspecial computing software which is loaded into a dedicated signalprocessor or onto a conventional computer.

Throughout, it has been assumed that, depending on the flow speed, thetransit time of a microparticle is 100 microseconds. Since themicroparticles arrive randomly to the laser beam, and because acombination of two particles or more is not allowed, the countingfrequency of the microparticles can at most be 1000 microparticles/s.If, for example, 10000 microparticles of each kind are to be counted andif there are 10 different kinds, then altogether it would take 100 s tomeasure one sample.

Examples of Performance and Embodiment of the Invention

Example 1

The set-up shown in FIG. 1 was tested in single-photon excitationconfocal fluorescence mode with a test-sample of Rhodamine-B dissolvedin water in a predetermined concentration. A continuous wave frequencydoubled Nd:YAG laser producing 532 nm wavelength was used as a lightsource. The illumination light was focused to the sample (3) with amicroscope objective (2b) with numerical aperture of 0.7. The sample (3)was placed in a position adjustable capillary tube (4) in conjunctionwith a simple liquid handling system. The emitted fluorescence light wasseparated from the illumination light by a dichroic filter (5) and apinhole (6), and detected by a photomultiplier tube (9). The limit ofdetection was 50 molecules in the focal volume. The sensitivity waslimited mainly by the background fluorescence and scattering from theoptical components. The dynamic range of the confocal measurement wasestimated to be about 3-4 orders of magnitude--the upper end was limitedby the maximum concentration of the dye before self-quenching in thesample becomes significant.

Example 2

The measurement was performed in two-photon excitation mode with aQ-switched (pulsed) Nd:YAG laser operating at 1064 nm wavelength and 20kHz repeat rate. In this test the photo-detector dark current was asignificant source of background noise but a cross-correlation circuitreduced this background by a factor of 100. The rest of background wascaused by second harmonic generation of 532 nm photons at the focalpoint, but this was suppressed using appropriate filters. The limit ofdetection was 1 molecule in the focal volume without cross-correlationand without second harmonic suppression filters. In two-photonexcitation self-quenching does not play any role. The dynamic range ofthis test instrument was 5 orders of magnitude and was estimated to bebetter than 8 orders of magnitude with proper filtering and thecross-correlation system. The results are shown in FIG. 5 which showsthe single photon count rate as a function of number of molecules in thefocal volume.

As it has become clear from these test measurements, it is feasible todetect single molecules tagged to the microparticles. On a 1 μmmicroparticle it is possible to attach 10⁶ antibodies. If 10% areactive, the dynamic range of the assay could be 10⁵ even when only asingle particle is used as a representative for a particular analyte.

The method and related device is subject to a statistical measurementerror which may be significant if the test result is based on only onemicroparticle. The imprecision is caused by the following factors, forexample: the microparticles have a limited monodispersity, the liquidhandling system does not provide precise hydrodynamic focusing, thecount rate from one microparticle is very limited at lowestconcentrations of the analyte. For the purpose of improving thestatistical precision of the method it is necessary to measure a largenumber of microparticles representing different analytes. As it has beendiscussed above in this text, averaging the results from 1000 or 10000microparticles ensures sufficient precision in a reasonable time.

As described above, identification of the category can be made usingfluorescent indicators D_(k). It also important that the signal fromthese indicators does not interfere with the signal obtained from thelabel F. Spectral filtering does not ensure sufficient separation of thesignals obtained from F and D_(k). This is particularly true if thefluorescence emission spectra overlap and the signals cover a largedynamic intensity range. For reducing these problems we introduceexamples of optical set-ups which ensure sufficient separation.

Example 3

With reference to FIG. 6, we assume the use of e.g. phycoerythrin asfluorescent label and a set of tetrapyrrolic dyes as identificationindicators D_(k). This concept provides very good separation of signalsbecause, according to our experiments, tetrapyrrolic dyes show very lowsusceptibility to two-photon excitation. Phycoerythrin, however, showsvery high susceptibility to two photon excitation. Therefore the idea ofthis example is to use two photon excitation at 1064 nm forphycoerythrin and single photon excitation at 532 nm for tetrapyrrolicdyes. In the instrumental set-up applicable to this example, thefundamental 1064 nm beam from a pulsed Q-switched Nd:YAG laser (14) isfrequency doubled in a crystal (15). The 1064 nm and the frequencydoubled 532 nm wavelength within the same beam is spit in two beams by adichroic mirror (16). The 532 nm beam is conducted through a long fibre(17) thus delaying it by e.g. 500 ns and then combined again with the1064 nm beam in the dichroic mirror (16). The light pulses at 1064 nmand 532 nm appear with 500 ns intervals and are focused to same focalpoint (22) in the capillary cuvette (23) with lenses (19), (20) and(21a). The set-up is equipped with an appropriate number of photondetectors. For simplicity, only two detectors (28) and (29) are shown inFIG. 6. The emission from the sample is collected by the objective lens(21a) and dichroic mirrors (24) and (25) and lenses (26a) and (26b)through pinholes (27a) and (27b) to detectors (28) and (29) which areconnected to an appropriate signal analysis system. In this set-up, theidentification signal D_(k) and the signal F appear at different times,which are separated by the delay time of 500 ns. The signal analysis issynchronized with the pulse laser and the temporal separation eliminatesany possible interference between F and D_(k).

Example 4

In this example we assume the use of e.g. phycoerythrin as fluorescentlabel and a set of cyanine dyes as identification indicators D_(k). Theidea of this example is to use two photon excitation at 1064 nm forphycoerythrin and single photon excitation at 630 nm for tetrapyrrolicdyes. Referring to FIG. 7 the instrumental set-up applicable for thisexample comprises a pulsed Q-switched Nd:YAG laser (31) with 1064 nmbeam used for excitation of phycoerythrin. The system comprises anotherlaser, e.g. a pulsed diode laser at 630 nm (32). The beams of theselasers are combined using a dichroic mirror (33) and then focused to thesame focal point (34) in the capillary cuvette (35) with lenses (36),(37) and (38). The set-up is equipped with an appropriate number ofphoton detectors. In FIG. 7 only two detectors (39) and (40) are shownfor simplicity. The emission from the sample is collected by theobjective lens (38), dichroic mirrors (41) and (42) and lenses (43a) and(43b) through pinholes (44a) and (44b) to detectors (40) and (39) whichare connected to appropriate signal analysis system. In this set-up, theidentification signal D_(k) and the signal F appear at different timesbecause the pulse lasers are activated at different times respectively.The signal analysis is synchronized with the lasers and the temporalseparation eliminates the possible interference between F and D_(k). Thelong shift between the emission bands of phycoerythrin and cyanine dyesfurther enhances the separation.

Example 5

In this example we assume the use of e.g. phycoerythrin as fluorescentlabel and identification indicators D_(k) based on Raman scattering. Theidea of this example is to use two photon excitation at 1064 nm forphycoerythrin and to detect Raman scattering over the 1100-1200 nmrange. Referring to FIG. 6, the instrumental set-up applicable for thisexample comprises a pulsed or CW Nd:YAG laser (14) producing a 1064 nmbeam. (The components 16, 17 and 18 shown in FIG. 6 are not applicablein this Example). The 1064 nm beam is focused to the sample (22) in thecapillary cuvette (23) with lenses (19), (20) and (21a). This set-upneeds a photon detector (28) for fluorescence emission from label F. Theemission from the sample is collected by the objective lens (21a) anddichroic mirrors (25) and lens (26a) through the pinhole (27a) to thedetector (28) which are connected to an appropriate signal analysissystem. In addition, a detection system for Raman scattering is needed.Raman detection can take place either by a separate 90 degree objectivelens (21b) or by the same objective lens (21a) as used for fluorescence.In both cases, Raman scattering will be focused to a pinhole (27b) or(27c) and detected with an appropriate Raman detector (29) or (30).Identification of the microparticle category on the basis of its Ramanspectrum can be performed using several alternative and commonly knowndetection methods. The spectral features specific for microparticles indifferent categories can be registered with several photon detectorswith appropriate spectral filters. Alternatively, registration of thespectrum or its interferogram (the Fourier transform of the frequencyspectrum) can be made using e.g. a conventional grating spectrometer oran interferometer, which are coupled to an array detector providingcontinuous spectrum with adequate resolution. The concentration of theindicators in the microparticle polymer can be high. The intensity ofthe Nd-YAG-laser is high enough to produce a signal in a short time andstrong enough for reliable identification. The identification of thecategory, i.e. correlation of spectral features, can be performed veryfast and simply using known correlation calculation methods.

Example 7

With the reference to FIG. 6 we assume the use of e.g. a coumarinderivative as fluorescent label F and a set of tetrapyrrolic or cyaninedyes as identification indicators D_(k). The idea of this example is touse two photon excitation at 820 nm for coumarin and single photonexcitation at 410 nm for tetrapyrrolic dyes. The instrumental set-upapplicable to this example is similar with that used in Example 3,except the laser which in this example is a diode pumped CrLi-Sapphirelaser with 80 MHz pulse rate, 30 fs pulse width, 0.5 nJ pulse energy andadjustable 820-900 nm wavelength. The laser is connected to a frequencydoubling crystal for the 410 nm line and to a fibre delay line. The highpulse frequency prerequisites lead to shorter delay times and fastersingle photon counting. In this set-up, the identification signal D_(k)and the signal F appear at different times which are separated by thedelay time. The signal analysis is synchronized with the laser pulsesand the temporal separation eliminates any possible interference betweenF and D_(k).

Example 8

In this example we assume the use of a polymethinecyanine dye, forexample BHDMAP (L. G. Lee & al. Cytometry 21(1995)120-128) asfluorescent label F and a set of tetrapyrrolic dyes as identificationindicators D_(k). The idea of this example is to use two separate lasersand single photon excitation. Referring to FIG. 7, the instrumentalset-up applicable for this example comprises a CW frequency doubledNd:YAG laser (31) with 532 nm beam for D_(k) and another laser, e.g. adiode laser at 785 nm (32) for excitation of F. Otherwise, the set-upfollows the same principles as in Example 4. The long shift between theemission bands of phycoerythrin and the cyanine dye enhances theseparation.

A specialist in the field appreciates that the different applications ofthe said invention may vary within the scope of the claims to bepresented in the following section. It will be appreciated that themethods of the present invention can be incorporated in the form of avariety of embodiments, only a few of which are disclosed herein. Itwill be apparent to the artisan that other embodiments exist and do notdepart from the spirit of the invention. Thus, the described embodimentsare illustrative and should not be construed as restrictive.

We claim:
 1. A biospecific multiparameter assay method based on the useof different categories of microparticles as solid phase representingdifferent analytes to be assayed, each category of microparticles beingcoated with a different primary biospecific reactant, wherein to saidmicroparticles has been added or the molecular structure of saidmicroparticles contains one or more florescent indicators D_(k) in oneor several concentrations, and the use of secondary biospecificreactants labelled with a photoluminescent label F, said methodcomprising the steps ofpooling the different categories ofmicroparticles together in a suspension and adding the sample containinganalytes to be assayed into the suspension, adding a mixture ofsecondary biospecific reactants labelled with the photoluminescent labelF into the suspension to initiate biospecific reactions between theanalytes and the labelled reactants and reactants bound to themicroparticles, diluting the suspension to reduce the concentration oflabelled reactants not bound to the microparticles, activating theindicators D_(k) and the photoluminescent label F, and measuring thephoton emission from the indicators D_(k) for the identification of themicroparticle category and the photon emission from the label F fordetermination of the analyte concentration, wherein the photoluminescentlabel F is a short decay time fluorescent label and the excitation anddetection of the label F is performed with two-photon excitation andthat the susceptibility to two-photon excitation of the fluorescentindicator D_(k) is substantially lower than that of the photoluminescentlabel F.
 2. The method according to claim 1 wherein the fluorescentindicator D_(k) comprises a set of fluorescent dyes including one ormore of tetrapyrrolic or cyanine compounds wherein said tetrapyrroliccompounds and said cyanine compounds have essentially the sameexcitation wavelength and separate, narrow and spectrometricallyresolvable fluorescence emission bands.
 3. The method according to claim2, wherein said tetrapyrrolic compounds are selected from the groupconsisting of porphyrin, chlorin, bacteriochlorin, purpurin,pheophorbide, phthalocyanine and naphthalocyanine.
 4. The methodaccording to claim 3 wherein the tetrapyrrolic compounds are selectedfrom the group consisting of 1) deuteroporphyrine IX, 2) mesoporphyrineIX, 3) proto-porphyrine (IX) dimethyl ester, 4) octaethyl-porphin, 5)tetraphenylporphin, 6) tetra-(2-methoxy)-phenylporphin, 7) chlorin ofcoproporphyrin dimethyl ester, 8) bacteriochlorin of coproporphyrindimethyl ester, 9) aluminum phthalocyanine and 10) zinc phthalocyanine.5. The method according to claim 2 wherein the indicator D_(k) is a setof fluorescent dyes including at least one cyanine compound selectedfrom the group consisting of3,3'-diethylthiatricarbocyanine perchlorate;1,1'-diethyl-2,2,'-dicarbocyanine iodide; 3,3'-diethyloxadicarbocyanineiodide; 3,3'-diethyloxatricarbocyanine iodide;3,3'-diethylthiadicarbocyanine iodide; 3,3'-diethylthiatricarbocyanineiodide; IR-125; IR-132; IR-140; and IR-144.
 6. The method according toclaim 1 wherein the fluorescent indicator D_(k) comprises a set offluorescent dyes including one or more of tetrapyrrolic or cyaninecompounds, and wherein the photoluminescent label F is phycoerythrinwherein said tetrapyrrolic compounds and said cyanine compounds haveessentially the same excitation wavelength and separate; narrow andspectrometrically resolvable fluorescence emission bands.
 7. The methodaccording to claim 6, wherein said tetrapyrrolic compounds are selectedfrom the group consisting of porphyrin, chlorin, bacteriochlorin,purpurin, pheophorbide, phthalocyanine and naphthalocyanine.
 8. Themethod according to claim 7, wherein the tetrapyrrolic compounds areselected from the group consisting of 1) deuteroporphyrine IX, 2)mesoporphyrine IX, 3) proto-porphyrine (IX) dimethyl ester, 4)octaethyl-porphin, 5) tetraphenylporphin, 6)tetra-(2-methoxy)-phenylporphin, 7) chlorin of coproporphyrin dimethylester, 8) bacteriochlorin of coproporphyrin dimethyl ester, 9) aluminumphthalocyanine and 10) zinc phthalocyanine.
 9. The method according toclaim 6 wherein the indicator D_(k) is a set of fluorescent dyesincluding one or more cyanine compounds selected from the groupconsisting of3,3'-diethylthiatricarbocyanine perchlorate;1,1'-diethyl-2,2,'-dicarbocyanine iodide; 3,3'-diethyloxadicarbocyanineiodide; 3,3'-diethyloxatricarbocyanine iodide;3,3'-diethylthiadicarbocyanine iodide; 3,3'-diethylthiatricarbocyanineiodide; IR-125; IR-132; IR-140; and IR-144.
 10. The method according toclaim 1, wherein said photoluminescent label F is a short decay timefluorescent label having a decay time of a few nanoseconds.
 11. Abiospecific multiparameter assay method based on the use of differentcategories of microparticles as solid phase representing differentanalytes to be assayed, each category of microparticles being coatedwith a different primary biospecific reactant, wherein to saidmicroparticles has been added or the molecular structure of saidmicroparticles contains one or more indicators D_(k) in one or severalconcentrations, and the use of secondary biospecific reactants labelledwith a photoluminescent label F, said method comprising the stepsofpooling the different categories of microparticles together in asuspension and adding the sample containing analytes to be assayed intothe suspension, adding a mixture of secondary biospecific reactantslabelled with the photoluminescent label F into the suspension toinitiate biospecific reactions between the analytes and the labelledreactants and reactants bound to the microparticles, diluting thesuspension to reduce the concentration of labelled reactants not boundto the microparticles, activating the indicators D_(k) and thephotoluminescent label F; and measuring the photon emission from theindicators D_(k) for the identification of the microparticle categoryand the photon emission from the label F for determination of theanalyte concentration wherein the photoluminescent label F is a shortdecay time fluorescent label and the excitation and detection of thelabel F is performed either with two-photon excitation or with aconfocal optical set-up, and wherein the indicators D_(k) are compoundsadded into the microparticle or a molecule structure of themicroparticle, said compounds or molecular structure being identifiableon the basis of their Raman spectra.
 12. The method according to claim11, wherein said photoluminescent label F is a short decay timefluorescent label having a decay time of a few seconds.